The present invention relates to a process of expressing and isolating recombinant proteins and recombinant protein products from plants, plant derived tissue or cultured plant cells, which process exploits (i) the high affinity between cellulose binding peptides and cellulose; (ii) the inherent abundance of cellulose in planta; and/or (iii) the simplicity associated with cellulose isolation from plants, plant derived tissue and cultured plant cells.
More particularly, the present invention relates to a process expressing and isolating recombinant proteins and recombinant protein products from plants, plant derived tissue or cultured plant cells, which process employs the expression of a fusion protein including a recombinant protein and a cellulose binding peptide fused thereto, plant homogenization, isolation of a fusion protein cellulosic matter complex and optional subsequent isolation of the fusion protein and/or the recombinant protein from the complex. The present invention further relates to nucleic acid molecules and to genetically modified or viral infected plants or plant cells which are useful while implementing the process, and further to a novel composition of matter which results from the process.
Citation or identification of any reference in this section or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
With the advent of recombinant technology, techniques for the genetic transformation of various host organisms, such as bacteria, yeasts, fungi, plants and animals, for the purposes of producing specific proteins through the expression of heterologous or foreign genes have been extensively developed.
Using these recombinant techniques and hosts, numerous commercially important recombinant proteins (examples of which are included hereinbelow) have been expressed and purified. Expression and isolation of a protein of interest on a commercial scale, neccesitate the selection of a suitable expression host. This selection largely depends on the economics of production and purification, as well as the ability of the host to accomplish the post-translational modifications needed for full biological activity of the recombinant protein.
Much of the early work in biotechnology was directed toward the expression of recombinant or "heterologous" proteins in prokaryotes like Escherichia coli and Bacillus subtilis because of the relative ease of genetic manipulation, growth in batch culture and large-scale fermentation of prokaryotes.
Although E. coli can in certain cases perform some post translational modifications and events, such as, protein folding and disulfide bond formation, it cannot secrete proteins extracellularly nor can it glycosylate, gamma carboxylate, beta hydroxylate, acetylate or process pre- and pro- peptides. B. subtilis suffers from the same limitations as E. coli except that it is capable of extracellular secretion.
Furthermore, E. coli and other bacteria are pathogens and therefore, depending on the application, contaminants such as pyrogens and endotoxins expressed along with the recombinant protein must be removed In addition, extensive post-purification chemical and enzymatic treatments (e.g., to refold the protein into an active form) are sometimes required in order to obtain a biologically active protein.
Because proteins are not secreted from prokaryotes like E. coli, bacterial cells must be disrupted for product recovery. The subsequent release of bacterial contaminants and other proteins make product purification more difficult and expensive. Because purification accounts for up to 90% of the total cost of producing recombinant proteins in bacteria, proteins like Insulin can cost several thousand dollars per gram when recombinantly produced in, and subsequently isolated from, E. coli.
Because of the many limitations associated with prokaryotic hosts, the biotechnology industry has looked for eukaryotic host cultures such as, yeast, fungi, insect cells, and mammalian cell tissue culture, to properly and efficiently express recombinant proteins.
For most of the proteins requiring extensive post-translational modifications for therapeutic and/or functional activity, mammalian cell culture is the most common alternative to E. coli. Although mammalian cells are capable of correctly folding and glycosylating bioactive proteins, the quality and extent of glycosylation can vary with different culture conditions among the same host cells. Furthermore, mammalian culture has extremely high fermentation costs (60-80% of total production expense), requires expensive media, and poses safety concerns from potential contamination by viruses and other pathogens. Yields are generally low and in the range of 0.5-1.5% of cellular protein, or micrograms per liter (up to 300-400 milligrams per liter).
Yeast, other fungi, and insect cells are currently being used as alternatives to mammalian cell culture. Yeast, however, produces incorrectly glycosylated proteins that have excessive mannose residues and are generally limited in eukaryotic processing. Further, although the baculovirus insect cell system can produce high levels of glycosylated proteins, these are typically not secreted, making purification complex and expensive. Fungi represent the best current system for high-volume, low-cost production of recombinant proteins, but they are not capable of expressing many target proteins.
In addition, eukaryotic cultures, require the maintenance of suitable conditions for efficient commercially viable expression of proteins. As such, the ambient temperature, pH value and aeration level of such cultures need to be carefully controlled, while nutrients must be added to the culture medium in carefully regulated doses and waste products removed. In addition, rigorous aseptic practices must be observed in order to avoid contamination by extraneous microbes. Such cultures are thus normally grown in sophisticated fermentors or bioreactors which are housed in expensively maintained factories. Such overheads are reflected in the high price of the recombinant protein end-products.
To a lesser extent, animals have also been utilized for the production of recombinant proteins. Although large amounts of protein can be produced and relatively easily recovered from such animals (e.g., proteins specifically produced in mammary glands and secreted with the milk), production in such host is limited to the expression of proteins which do not interfere with the host physiology. In addition, transgenic animals are subject to lengthy lead times to develop herds with stable genetics, high operating costs, contamination by animal viruses and a relatively slow rate of biomass generation substantially prolonging the time period following which recovery of commercial amounts of the protein can be effected.
The biochemical, technical and economic limitations on existing prokaryotic and eukaryotic expression systems has created substantial interest in developing new expression systems for the production of recombinant proteins.
Plants represent the most likely alternative to existing expression systems. With the availability and on going development of plant transformation techniques, most commercially important plant species can now be genetically modified to express a variety of recombinant proteins.
Such transformation techniques include, for example, the Agrobacterium vector system, which involves infection of the plant tissue with a bacterium (Agrobacterium) into which the foreign gene has been inserted. A number of methods for transforming plant cells with Agrobacteriumn are well known (Klee et al., Annu. Rev. Plant Physiol. (1987) 38:467-486; Schell and Vasil Academic Publishers, San Diego, Calif. (1989) p. 2-25; and Gatenby (1989) in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. p. 93-112).
The biolistic or particle gun method, which permits genetic material to be delivered directly into intact cells or tissues by bombarding regeneratable tissues, such as meristems or embryogenic callus, with DNA-coated microparticles has contributed to plant transformation simplicity and efficiency. The microparticles penetrate the plant cells and act as inert carriers of a genetic material to be introduced therein. Microprojectile bombardment of embryogenic suspension cultures has proven successful for the production of transgenic plants of a variety of species. Various parameters that influence DNA delivery by particle bombardment have been defined (Klein et al., Bio/Technology (1998) 6:559-563; McCabe et al., Bio/Technology (1998) 6:923-926; and Sanford, Physiol. Plant. (1990) 79:206-209).
Micropipette systems are also used for the delivery of foreign DNA into plants via microinjection (Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; and Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217).
Other techniques developed to introduce foreign genes into plants include direct DNA uptake by plant tissue, or plant cell protoplasts (Schell and Vasil (1987) Academic Publishers, San Diego, Calif. p. 52-68; and Toriyama et al., Bio/Technology (1988) 6:1072-1074) or by germinating pollen (Chapman, Mantell and Daniels (1985) W. Longman, London, p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719).
DNA uptake induced by brief electric shock of plant cells has also been described (Zhang et al., Plant. Cell. Rep. (1988) 7:379-384 and Fromm et al., Nature (1986) 319:791-793).
In addition, virus mediated plant transformation has also been extensively described. Transformation of plants using plant viruses is described, for example, in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693, EPA 194,809, EPA 278,667, and Gluzman et al., (1988) Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189. Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, have also been described, for reference, see, for example WO 87/06261.
The production of recombinant proteins and peptides in plants has been investigated using a variety of approaches including transcriptional fusions using a strong constitutive plant promoter (e.g., from cauliflower mosaic virus, Sijmons et al., Bio/Technology (1990) 8:217-221); transcriptional fusions with organ specific promoter sequences (Radke et al., Theoret. Appl. Genet. (1988) 75:685-694); and translational fusions which require subsequent cleavage of a recombinant protein (Vanderkerckove et al., Bio/Technology (1989) 7:929-932).
The application of such genetic transformation techniques has allowed the incorporation of a variety of important genetic traits for crop improvement and also for the biotechnological production of extractable, valuable, foreign proteins including enzymes, vaccine proteins and antibodies.
Foreign proteins that have been successfully expressed in plant cells include proteins from bacteria (Fraley et al. Proc. Natl. Acad. Sci. U.S.A (1993) 80:4803-4807), animals (Misra and Gedamu, Theor. Appl. Genet. (1989) 78:161-168), fungi and other plant species (Fraley et al. Proc. Natl. Acad. Sci. U.S.A. (1983) 80:4803-4807). Some proteins, predominantly markers of DNA integration, have been expressed in specific cells and tissues including seeds (Sen Gupta-Gopalan et al. Proc. Natl. Acad. Sci. U.S.A. (1985) 82:3320-3324; Radke et al. Theor. Appl. Genet. (1988) 75:685-694).
Due to the advantageous economics of field-grown crops, the ability to synthesize proteins in storage organs like tubers, seeds, fruits and leaves and the ability of plants to perform many of the post-translational modifications previously described, several plant expression systems are currently investigated for potential as highly effective and economically feasible systems for the production of recombinant proteins.
Since highly expressive systems such as the ubiquitin fusion system described in U.S. Pat. No. 5,773,705 have been demonstrated, a major hurdle to an effective plant expression system resides with the relatively complicated purification procedures necessary in order to purify the recombinant protein.
As such, alternative expression approaches have been undertaken in an effort to simplify the purification procedure of the recombinant protein from the plant cells.
One such system focuses on the use of seed-storage protein promoters as a means of deriving seed-specific expression. Using such a system, Vanderkerckove et al., (Bio/Technol. (1989) 7:929-932) expressed the peptide Leu-enkephalin in seeds of Arabidopsis thaliana and Brassica napus. The level of expression of this peptide was quite low and it appeared that expression of this peptide was limited to endosperm tissue.
Another system utilizing seeds as an expression host is disclosed in U.S. Pat. No. 5,888,789. This system provides for the secretion of heterologous protein by malting of monocot plant seeds. The heterologous genes are expressed during germination of the seeds and isolated from a malt.
U.S. Pat. No. 5,580,768 describes a method of producing a genetically transformed fluid-producing plant. The genetically transformed plant which can be for example, a rubber secreting (Hevea) plant is capable of expressing the target product in the fluid that it produces which in this case is latex.
U.S. Pat No. 5,650,554 describes the use of a class of genes called oil body protein genes, that have unique features, allowing the production of recombinant proteins that can be easily separated from other host cell components.
Many additional expression systems have been described utilizing specific targeting or directing of recombinant proteins to specific plant tissues.
Although systems which target or direct recombinant protein production to specific tissues allow for easier recombinant protein isolation such systems are typically limited in the effective host range and/or the amounts of recombinant proteins produced since such systems fail to exploit the entire plant biomass.
A novel approach for simplifying the purification of recombinant enzymes from plant host cells is disclosed in U.S. Pat. No. 5,474,925 which describes an expression construct utilizing a signal peptide translationally fused to a recombinant enzyme which targets the enzyme to the cellulose matrix of the cell wall. This enables the isolation of the enzyme along with the easily recoverable cellulose matrix. This system is utilized for the localized expression of commercially important enzymes in cotton fibers. According to this system, the expressed enzymes are recovered along with the cellulosic matter of the fibers. The enzyme-cellulose matrix recovered, is directly utilized for commercial enzymatic processes.
Although this system presents a simple means with which a recombinant protein can be expressed and isolated, it is limited to the production of enzymes in cotton fibers of the cotton plant.
Furthermore, a major hurdle encountered when expressing cellulose targeted proteins within a plant is the interference of the expressed products in the natural formation of the cell wall, which typically results in growth arrest of the plant growth. Although this hurdle can be overcome by, for example, targeting the protein to specific plant tissue as is the case for U.S. Pat. No. 5,474,925, this targeting severely limits the expressing biomass and as such the quantity of the expressed protein. In addition, targeting the expression to a specific plant tissue also limits the number of plant species which can be effectively utilized for such an expression.
There is thus a widely recognized need for, and it would be highly advantageous to have, a plant expression system and method which provide high level of expression of a recombinant protein and which allow simple and effective recovery of the expressed recombinant protein devoid of the above limitations.